Role of the gerI Operon of Bacillus cereus 569 in the Response of Spores to Germinants

Bacillus cereus 569 (ATCC 10876) germinates in response to inosine or to l-alanine, but the most rapid germination response is elicited by a combination of these germinants. Mutants defective in their germination response to either inosine or to l-alanine were isolated after Tn917-LTV1 mutagenesis and enrichment procedures; one class of mutant could not germinate in response to inosine as a sole germinant but still germinated in response to l-alanine, although at a reduced rate; another mutant germinated normally in response to inosine but was slowed in its germination response to l-alanine. These mutants demonstrated that at least two signal response pathways are involved in the triggering of germination. Stimulation of germination in l-alanine by limiting concentrations of inosine and stimulation of germination in inosine by low concentrations of l-alanine were still detectable in these mutants, suggesting that such stimulation is not dependent on complete functionality of both these germination loci. Two transposon insertions that affected inosine germination were found to be located 2.2 kb apart on the chromosome. This region was cloned and sequenced, revealing an operon of three open reading frames homologous to those in the gerA and related operons of Bacillus subtilis. The individual genes of this gerI operon have been named gerIA, gerIB, and gerIC. The GerIA protein is predicted to possess an unusually long, charged, N-terminal domain containing nine tandem copies of a 13-amino-acid glutamine- and serine-rich sequence.Bacillus species have the ability, under certain nutrient stresses, to undergo a complex differentiation process resulting in the formation of a highly resistant dormant endospore (6). These spores can then persist in the environment for prolonged periods until a sensitive response mechanism detects specific environmental conditions, initiating the processes of germination and outgrowth (9, 21, 25). Germination can be initiated by a variety of agents (12), including nutrients, enzymes, or physical factors, such as abrasion or hydrostatic pressure.The molecular genetics of spore germination has been most extensively studied in Bacillus subtilis 168 (21). B. subtilis spores can be triggered to germinate in response to either l-alanine or to a combination (29) of asparagine, glucose, fructose, and potassium ions (AGFK). Mutants of B. subtilis which are defective in germination responses to one or to both types of germinant have been isolated previously (20, 27). Analysis of these mutants suggests that the germinants interact with separate germinant-specific complexes within the spore (21). This in some way leads to activation of components of the germination apparatus common to both responses, such as germination-specific cortex lytic enzymes, leading in turn to complete germination of the spore (10, 22). The mutations within the gerA operon of B. subtilis specifically block germination initiated by l-alanine (34). The predicted amino acid sequences of the three GerA proteins encoded in the operon suggest that these proteins could be membrane associated, and they are the most likely candidates to represent the germinant receptor for alanine (21).The amino acid l-alanine has been identified as a common but not universal germinant in a variety of Bacillus species, often requiring the presence of adjuncts such as electrolytes and sugars. Ribosides, such as inosine, represent another type of common germinant, although many species are unable to germinate rapidly in response to these without the addition of l-alanine (9).The food-borne pathogen Bacillus cereus is a major cause of food poisoning of an emetic and diarrheal type (13, 16). The germination and growth of Bacillus cereus spores during food storage can lead to food spoilage and the potential to cause food poisoning (16). B. cereus has been shown to germinate in response to l-alanine and to ribosides (11, 18, 23). Spore germination can be triggered by l-alanine alone, but at high spore densities this response becomes inhibited by d-alanine, generated by the alanine racemase activity associated with the spores (8, 11). This auto-inhibition of l-alanine germination can be reduced by the inclusion of a racemase inhibitor (O-carbamyl-d-serine) with the germinating spores (11).Inosine is the most effective riboside germinant for B. cereus T, while adenosine and guanosine are less potent (28). The rate of riboside-triggered germination has been reported to be enhanced dramatically by the addition of l-alanine (18). It is unclear whether ribosides can act as a sole germinant, or whether there is an absolute requirement for l-alanine (28).An attempt has been made to analyze genetically the molecular components of the germination apparatus in B. cereus in order to dissect the germination responses of this species and to determine whether riboside-induced germination involves components related to those already described for amino acid and sugar germinants in B. subtilis.